The overall goal of the four interdependent projects in this Program Project is to carry out functional genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has become a model for the assemblage of over 250,000 species of non-yeast fungi. Most Neurospora genes have no homologs in yeasts and nearly 40% have no strong homologs in any organism to date, suggesting that examination of the functions of these genes will both novel and informative. Neurospora is an important model for basic research in eukaryotes, and fungi allied to Neurospora include significant animal and plant pathogens and industrial strains yielding antibiotics, chemicals, enzymes, and pharmaceuticals. The 43 Mb Neurospora genome is completely sequenced (>16 fold archival coverage), and automated annotation using programs trained on Neurospora genes predict 10,082 proteins. The first Project will pursue the systematic disruption of genes through targeted gene replacements, preliminary phenotyping of these strains, and their distribution to the scientific community at large. Through a primary focus in Annotation and Genomics, we will produce a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes, relying on data from EST analyses to refine the gene structures. Oligonucleotide-based microarrays will allow Transcriptional Profiling of the nearly 11,000 distinguishable transcripts in Neurospora. This effort will provide a baseline analysis of gene expression under a variety of growth conditions, and later begin to analyze the global effects of loss of novel genes; these data will be made available through the web via structures. Since alternative splicing, alternative promoters, and long antisense transcripts contribute widely to the overall complexity of expressed sequences in Neurospora, cDNA libraries will be generated from wild type and related strains to document this complexity to aid in annotation. Sequences from related strains will drive assembly of a SNP map. Overall, this effort will help to anchor genomic exploration within the largely unexplored phylogenetic Kingdom of the Fungi.